2.13. Tumour microarray analysis

IHC staining of the tumour microarray (TMA) was analysed using QuPath software version 0.3.2 (https://qupath.github.io/) (29). Cells were automatically detected, and tumour areas annotated. Thresholds for nuclear DAB staining were set at optical density (OD) 0.1, 0.3 and 0.5 to determine negative, weak, moderate and strong staining in individual cells. Histoscores for each tumour core were generated based on the proportion of cells classified within each of these categories automatically within QuPath. Evaluable tumour cores were available for 231/365 cases. Where available, triplicate (54/231) and duplicate (84/231) cores for each case were analysed, and mean values were used for statistical analysis. Duplicate and triplicate cores demonstrated good concordance between histoscores (median coefficient of variation 12.9%).